The activity spectrum and mode of action of native antibacterial peptides represent some of the most exciting subjects of the Millennium, mostly because of the rapid increase of bacteria that become resistant to conventional antibiotics, and the promise of native antibacterial peptides to combat these strains in the clinical setting. Many antibacterial peptides have intracellular target biopolymers, but because the peptides have to reach the cell interior, their action on bacterial and host cell membranes often mask their major mode of action. The best molecules to study these processes are the short, proline-rich antibacterial peptides originally isolated from insects. Pyrrhocoricin, drosocin and perhaps apidaecin appear to inhibit chaperone-assisted protein folding via binding to the multihelical lid region of the 70 kDa heat shock protein DnaK with their amino terminal halves. The carboxy-terminal domains are likely involved in the process of penetration into bacterial and host cells. A designed dimeric pyrrhocoricin analog is currently studied for its ability to protect mice against systemic and local Klebsiella pneumoniae, Escherichia coli, Haemophilus influenzae, Moraxella catarrhalis and Salmonella typhimurium infections. This lead compound cannot kill Staphylococcus aureus, Streptococcus pneumoniae, Helicobacter pylori or Haemophilus ducreyi, among other strains, in vitro. This grant application is concerned with the development of novel peptidic and non-peptidic Hsp70 inhibitors for later development as antibacterial, anti-fungal, anti-parasitic or insecticide agents. In the Phase I stage of this proposal, we will identify the role of each individual amino acid residue in pyrrhocoricin and drosocin in inactivating DnaK and in promoting bacterial or eukaryotic cell entry. These properties will be initially studied on peptides in which each native residue is replaced by alanine or tyrosine. The experiments involving DnaK will include binding studies to the recombinant protein and its synthetic DE helix fragment, the inhibition of the inherent ATPase activity of the protein, and, if inconclusive data are obtained, the inhibition of protein folding as indicated by the alkaline phosphatase and beta-galactosidase activity of E. coli cultures. The ability of the peptide analogs to enter E. coli cells and mouse macrophages will be studied by confocal fluorescence microscopy and scanning electron microscopy. In Phase II, we will identify the pyrrhocoricin-interacting residues in E. coli, H. influenzae and Agrobacterium tumefaciens DnaK by using spectroscopic and molecular modeling techniques as well as by fluorescence polarization studies between the peptides and mutated DnaK fragments. When the required structural framework for the two independent peptide functions is identified, a new generation of peptide analogs and peptidomimetic structures will be designed. To this end, the DnaK-interacting residues will be modified to bind to the 70 kDa heat shock protein of currently unresponsive bacterial and fungal strains as well as that of Drosophila modeling obnoxious insects. This line of investigations will include a peptide library with fixed delivery and variable DnaK-binding residues as well as de novo design of Hsp70 inhibitors. Measurements of the in vitro Hsp70-binding and antimicrobial activities will be complemented with a limited set of in vivo efficacy studies. It is our hope that by the end of the grant period we will be able to dissect the native antibacterial peptide sequences to functional domains with amino acid residue accuracy and will provide a new generation of peptide and peptidomimetics lead compounds to fight currently life-threatening infectious diseases and will present a new family of biopesticides.